The government may own certain rights in the present invention pursuant to NIH grant No. 177590.
1. Field of Invention
The present invention relates to methods and compositions for isolation of nucleic acids from cells. In particular aspects, this invention relates to the use of chaotropic compositions, such as guanidine hydrochloride or guanidinium isothiocyanate, in combination with polyanionic compositions, such as those containing sulfated polysaccharides (heparin), for the isolation of nucleic acids (DNA or RNA).
2. Description of the Related Art
Techniques for isolating nucleic acids from cells have been employed for many years. With the increasing importance of molecular biology, biochemistry, virology and cellular biology, along with the recent technological advances in these disciplines, the need for isolated quality nucleic acids has increased considerably. It is not sufficient to now merely ,isolate, nucleic acids prior to their utilization in different experiments. The isolation of nucleic acids is one important initial step in any protocol where the use of nucleic acids is envisioned, whether it be DNA or RNA. In fact, the success of an entire experimental protocol may lie in the initial steps of isolating quality nucleic acids.
Thus, within the last few years, it has been an aim of molecular biologists to identify isolation procedures for nucleic acids which yield quality material in acceptable quantities. An important `quality` of the isolated nucleic acid is that the product be essentially free of contaminating substances which might otherwise interfere with subsequent experimental manipulations. For example, the contaminating substances may be proteins or residual compounds or chemicals used during the isolation procedure.
It is particularly desirable to isolate nucleic acids that are relatively intact and thus not appreciably degraded. This is important where one seeks to obtain nucleic acids in excess of 10 kilobases. Nucleic acid isolation often precedes a tedious experimental protocol which, more than likely, will require extensive handling and manipulations of the nucleic acids. However, it is often difficult to isolate intact, high molecular weight DNA (size in excess of 15 kilobases) because the size of the DNA itself imposes inherent handling problems (1-3). With this information in mind, and in order to improve recovery of amount of nucleic acid isolated, as well as quality, handling and processing time for isolating nucleic acids are a concern when developing new protocols.
Once isolated, nucleic acids will typically need to be dissolved in a buffer of choice. If the isolated nucleic acids is not capable of being dissolved in the required buffer system, then even relatively intact and purified nucleic acids will not be useful. Difficulties in resuspending the isolated nucleic acid product have presented problems (4). Thus, nucleic acid isolation methods should reproducibly generate intact nucleic acids which are essentially free of contaminating substances, which will dissolve in a selective buffer and are, therefore, functional in a variety of different experimental designs (restriction enzyme analyses, cloning into specific vectors, mutating by point mutation, etc.).
Enzymes are often employed to assist in purifying nucleic acids free of associated macromolecules, such as protein, lipids, etc. However, the use of enzymes (pronase, RNAse, DNAse, etc.) to selectively eliminate one component involves a risk factor that is difficult to measure. For example, elimination of RNA from DNA samples will require the use of specially purified RNAse that are `essentially DNAse free`. Thus, a valuable sample of DNA is potentially at risk of becoming degraded where even minute amounts of DNAse remain in the RNAse.
Inactivating nucleases, inherent components of most cells, present another problem with which the investigator must deal with. When cells are disrupted, nucleases are released which will tend to degrade the nucleic acid sought to be isolated. A variety of denaturants (e.g., urea, SDS, guanidine hydrochloride and guanidinium isothiocyanate) have been employed with varying degrees of success to inactivate endogenous nucleases and proteases (5-11). Unfortunately, these agents alone have not been shown to provide isolated nucleic acids of the highest quality.
As noted, it is not uncommon to isolate nucleic acids which are degraded due to the extensive handling required by the individual protocol. The average size of DNA obtained from currently available isolation protocols is typically on the order of about 20-40 kilobases (2,3,7). A protocol which would eliminate some handling would thus offer an advantage over existing protocols. Also, a protocol which would allow for the isolation of even higher molecular weight nucleic acids (e.g., greater than 75 kilobases) would be useful for a variety of different experimental approaches (preparation of cosmid libraries, etc).
Organic solvents, such as phenol have also been utilized to aid in the elimination of proteins. Organic solvents are helpful in nucleic acid purification protocols, but present a tedious problem in terms of safety as well as in eliminating traces of remaining solvents. These solvents may retard the dissolution of the nucleic acid into an appropriate buffer as well as hinder further enzymatic manipulation of the nucleic acid (4,11).
In light of these and other drawbacks in the prior art for isolating nucleic acids, there is a need for an isolation method which is generally applicable to numerous cell types, as well as reproducible, efficient and inexpensive. The invention disclosed herein presents methods and compositions which allow for the timely, efficient, inexpensive and straightforward purification of nucleic acids without worry of degradation, elimination of wrong components, or producing a product which will not be functional in further experimentation. The invention described herein relates to the efficient purification of high molecular weight nucleic acids (often greater than 75 kilobases) which are relatively free of unwanted components, are essentially intact, are usually able to dissolve in an appropriate buffer system, and are thereby functional in a variety of experimental protocols ranging across many different disciplines of research.